Disposable system for analysis of hemostatic function

ABSTRACT

A disposable system, in some embodiments, includes a multi-channel or multi-chamber test cartridge device configured to operate with a testing system for evaluation of hemostasis in a subject by in vitro evaluation of a test sample from the subject. The disposable system, in some embodiments, is configured to interrogate the test sample to evaluate clot stiffness, strength, or other mechanical properties of the test sample to assess the function of various physiological processes occur during coagulation and/or dissolution of the resulting clot.

RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.15/958,875, filed Apr. 20, 2018, now U.S. Pat. No. 11,366,093, whichclaims priority to, and the benefit of, U.S. Provisional Appl. No.62/488,045, filed Apr. 20, 2017, titled “Disposable System for Analysisof Hemostatic Function,” each of which is incorporated by referenceherein in its entirety.

GOVERNMENT LICENSE RIGHTS

The invention was made with government support under grant R44HL103030awarded by National Heart Lung and Blood Institute. The government hascertain rights in the invention.

TECHNICAL FIELD

The present application relates to devices, systems and methods forevaluating hemostasis in a subject by preparation and analysis of a testsample from the subject.

BACKGROUND

Hemostasis, the physiological control of bleeding, is a complex processincorporating the vasculature, platelets, coagulation factors,fibrinolytic proteins, and a variety of activators and inhibitors.Disruption of hemostasis plays a central role in the onset of myocardialinfarction, stroke, pulmonary embolism, deep vein thrombosis andexcessive bleeding. Consequently, in vitro diagnostics (IVD) arecritically needed to quantify hemostatic function/dysfunction and directappropriate treatment.

The process of coagulation is highly dependent, among other things, onthe temperature at which it takes place. Under normal conditions,coagulation occurs at body temperature, which is optimal for the properenzymatic action of the clotting factors in the cascade.

Preparation of the blood to be tested is also important, as the manner ablood sample is prepared prior to its evaluation can affect, forexample, the actions of the vasculature components, platelets and othercellular components, coagulation factors, fibrinolytic components, andany inhibitor or activator of hemostasis.

SUMMARY

Provided are devices, systems and methods for evaluation of hemostasis.For example, provided are disposable systems for analysis of hemostasisfunction. The disposable system, in some embodiments, includes amulti-channel or multi-chamber test cartridge device configured tooperate with a testing system for evaluation of hemostasis in a subjectby in vitro evaluation of a test sample from the subject. The disposablesystem, in some embodiments, is configured to interrogate the testsample to evaluate clot stiffness, strength, or other mechanicalproperties of the test sample to assess the function of variousphysiological processes occur during coagulation and/or dissolution ofthe resulting clot. The sample can include in whole, or in part, wholeblood, plasma, platelet rich plasma, or platelet poor plasma.Furthermore, the sample can include one or more reagent (such asanticoagulants or anti-platelet drugs that might be present in the bloodas collected), or one or more pharmacological treatment (such as in thecase of heparin or low molecular weight heparin) or other inertcomponents (such as polystyrene beads) that are added to the test samplebefore the cartridge device being used. The disposable systemfacilitates the point of care evaluation of hemostasis of a test samplethat is robust (e.g., can be performed in non-laboratory environment),rapid (e.g., only to take a few minutes to perform), easy-to-use andprovides clear results (e.g., that are direct to the functionalcomponents of hemostasis), and facilitates identification of exacthemostasis defects. The exemplified device automates one or morepre-measurement steps that minimizes sample manipulation steps requiredfor the user, thereby improving test reproducibility and/or testquality. The disposable system, in some embodiments, includes aplurality of testing circuits each having a pathway defined by channelsand chambers configured to prepare a test sample of blood for evaluationby a measurement device. In each testing circuit, a portion of the testsample is introduced to a reagent or combination of reagents specific tothat testing circuit.

The disposable system, in some embodiments, is configured to conditionthe respective test samples prior to, during, and/or after the mixingwith the reagent(s), to optimize the proper actions of applicable bloodcomponent and chemistry (e.g., vasculature components, platelets orother cellular components, coagulation factors, fibrinolytic components,and any other inhibitor or activator of hemostatic function, etc.) beingevaluated.

In an aspect, an apparatus (e.g., a cartridge) is disclosed for theassessment of hemostasis. The apparatus includes a housing; an inputport integrally formed with the housing that is structurally configuredto establish fluidic communication and evacuate contents of a sampleholding tube; and a first chamber in fluidic communication with theinput port, the first chamber being configured to receive a samplecontained in the sample holding tube and to condition the receivedsample to a desired temperature (e.g., a pre-defined temperature range)before the received sample is allowed to contact one or more reagentslocated in one or more fluidic circuits downstream to the first chamber,wherein each of the one or more fluidic circuits comprises i) a secondchamber in fluidic communication with the first chamber that meters thesample in the first chamber into an aliquot, wherein the metered sampleis introduced to a reagent, or a combination of reagents, (e.g., in theform of lyophilized reagent bead) located in a corresponding fluidiccircuit (e.g., a reagent pocket) to form a mixed sample and ii) atesting chamber in fluidic communication with the second chamber, thetesting chamber being structurally configured for interrogation by ameasurement system configured to determine properties (e.g., mechanicalproperties or viscoelastic properties) of the mixed sample.

In some embodiments, at least one of the one or more fluidic circuitscomprise one or more pockets (e.g., each configured to house alyophilized reagent bead comprising a reagent, or a combination ofreagents).

In some embodiments, at least one of the one or more fluidic circuitscomprise one or more liquid-retaining pockets (e.g., each configured tohouse an assay, in liquid form, comprising the reagent, or a combinationof reagents).

In some embodiments, at least one of the one or more fluidic circuitsincludes one or more lyophilized reagents that are located on one ormore surfaces thereof (e.g., lyophilized on each of the surfaces;lyophilized as films placed on, or adhered to, one or more of thesurfaces).

In some embodiments, at least one of the one or more fluidic circuitsincludes one or more reagents that are processed onto surfaces thereof(e.g., dried on the surfaces; spray coated on the surfaces; baked ontothe surfaces).

In some embodiments, the input port is communicatively coupled to apressure port, wherein pressure applied to the pressure port causes theevacuation of the contents of the sample holding tube through the inputport to first chamber.

In some embodiments, the input port comprises a needle assembly.

In some embodiments, the needle assembly comprises the input port and asecond port, wherein the second port is configured to vent a liquid orgas into the sample holding tube so as to promote evacuation of thecontents therein. The input port, in some embodiments, is located (e.g.,concentrically located) within a second port configured to vent a liquidor gas into the sample holding tube so as to cause evacuation of thecontents of the sample holding tube.

In some embodiments, the input port comprises a luer lock configured toconnect to the sample holding tube, wherein the sample holding tube is asyringe.

In some embodiments, the input port is communicatively coupled to afirst pressure port, wherein pressure when applied to the first pressureport causes the evacuation of the contents of the sample holding tubethrough the input port to the first chamber.

In some embodiments, the first chamber is configured to mate with acorresponding thermal regulating system (e.g., heating/cooling system)of the measurement system to condition the received sample to, or near,the desired temperature.

In some embodiments, the shape and/or materials of the first chamber areoptimized to facilitate thermal regulation (e.g., heating and/orcooling) of the sample to, or near, the desired temperature.

In some embodiments, the first chamber is configured to mate with acorresponding thermal regulating surface of a sub-system component ofthe measurement system to condition the received sample to, or near, thedesired temperature. In some embodiments, a channel portion of the oneor more fluidic circuits is configured to mate with a correspondingheating/cooling system of the measurement system to condition thereceived sample to, or near, the desired temperature.

In some embodiments, a channel portion of the one or more fluidiccircuits is configured to mate with a corresponding thermal regulatingsystem of the measurement system to condition the received sample to thedesired temperature. In some embodiments, the first chamber and/or thechannel portion of the one or more fluidic circuits is in physicalproximity (e.g., physical contact or near contact) with a sensorconfigured to measure a temperature of the sample received in the firstchamber.

In some embodiments, the sensor is selected from group consisting of athermistor, a thermocouple, and an optical sensor (e.g., an IR sensor).

In some embodiments, the apparatus includes a first pressure port influidic communication with the first chamber, the first pressure portbeing configured to receive negative or differential pressure (e.g., forfilling the first chamber); and a filter positioned within the firstpressure port in at least one of the fluidic circuits (e.g., such thatthe filter is clogged by the sample received in the first chamber whenthe first chamber is full). The filter, in some embodiments, isconfigured to allow air to move through the first pressure port butprevent fluid from moving there through.

In some embodiments, the apparatus includes a first pressure portconfigured to receive negative or differential pressure for filling thefirst chamber; and a first fluidic pathway extending from the firstpressure port to the first chamber, wherein the filter is positionedwithin the first pressure port.

In some embodiments, for each of the one or more fluidic circuits, thefluidic communication between the first chamber and the second chamberis through a second fluid pathway originating from a side of the firstchamber (e.g., a side wall, a bottom wall, and etc.) (e.g., such thatbubbles present in the received sample are trapped away from the secondchamber).

In some embodiments, each of the one or more fluidic circuits comprisesa third fluid pathway in fluidic communication with the second chamber,wherein the third fluidic pathway leads a second pressure portconfigured receive negative or differential pressure for filling thesecond chamber.

In some embodiments, the second pressure port has a second filtertherein, wherein the second filter is configured to clog when the secondchamber is filled.

In some embodiments, the apparatus includes one or more fluidic pathwaysin fluidic communication with the second pressure port for all of theone or more fluidic circuits, wherein the one or more fluidic pathwaysare configured to provide the negative pressure to the second pressureport for all of the one or more fluidic circuits.

In some embodiments, for each of the one or more fluidic circuits, thesecond chamber is in fluidic communication with a vent port, wherein thevent port is configured to be closed while the sample is metered intothe aliquot in the second chamber and further configured to be open toatmospheric pressure after the sample is metered into the aliquot in thesecond chamber.

In some embodiments, each of the one or more fluidic circuits comprisesa third set of fluid pathways in fluidic communication between arespective second chamber (e.g., metering chamber) and test chamber,wherein a portion of third set of fluid pathways are arranged as aserpentine-shaped conduit or channel.

In some embodiments, each of the one or more fluidic circuits furthercomprises a serpentine reservoir between the testing chamber and thesecond chamber.

In some embodiments, the metered sample is alternatively directedthrough portions of the one or more fluidic circuits to facilitatemixing of the metered sample and the reagent, or a combination ofreagents.

In some embodiments, the metered sample is alternatively andmultiplicatively directed, for each of the one or more fluidic circuits,between a first position (e.g., the second chamber) in a fluidic circuita second position (e.g., a position in the serpentine reservoir) in thefluidic circuit.

In some embodiments, each of the one or more fluidic circuits furthercomprises a third pressure port in fluidic communication with the secondchamber and the testing chamber, the third pressure port configured toreceive negative or differential pressure (e.g., for drawing the aliquotfrom the second chamber to the testing chamber), wherein the thirdpressure port is further configured to alternately receive alternatingpressure, e.g., for alternately drawing the aliquot from the secondchamber along the serpentine reservoir and pushing the aliquot throughthe serpentine reservoir to the second chamber.

In some embodiments, the serpentine reservoir includes an opticaldetection zone to facilitate optical detection of the metered sample inthe serpentine reservoir or a location of the sample in the serpentinereservoir.

In some embodiments, each of the one or more fluidic circuits furthercomprises a mixing pathway between the testing chamber and the secondchamber, the mixing pathway comprising one or more ferromagnetic beadsor bars therein.

In some embodiments, at least one of the one or more fluidic circuitscomprises one or more quality testing portals.

In some embodiments, the one or more quality testing portals isconfigured to be sensed optically, wherein the quality testing port istransparent.

In some embodiments, the one or more quality testing portals isconfigured to be sensed electrically, wherein quality testing portcomprises one or more sensing electrodes.

In some embodiments, the one or more quality testing ports is configuredto be sampled for characteristics for the metered sample (e.g., forpressure, presence of flow, flow rate, temperature).

In some embodiments, for each of the one or more fluidic circuits, thetesting chamber comprises a mechanism to couple energy into the testingchamber to perform the measurements such as in the case of a lensconfigured to direct ultrasonic pulses into to the testing chamber.

In another aspect, an apparatus is disclosed for the assessment ofhemostasis, the apparatus comprising: a housing; an input portintegrally formed with the housing that is structurally capable ofestablishing fluidic communication with, and evacuating contents of, asample holding tube; a first chamber that is in fluidic communicationwith the input port that receives the sample contained in the evacuatedtube and whereby the sample temperature is adjusted to a desiredtemperature before the sample contacting one or more reagents; one ormore second chambers that are in fluidic communication with the firstchamber, the one or more second chamber being configured to meter thesample in the first chamber into one or more aliquots; one or morereagent pockets each filled with one or more lyophilized reagent beadthat are in fluidic communication with each of the aliquot chambers andpermits the sample present in each aliquot to be mixed with said one ormore reagent beads; and one or more testing chambers that are in fluidiccommunications with the aliquot chambers and that are structurallycapable of being interrogated to determine the sample viscoelasticproperties after such sample has been mixed with the one or morereagents.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes an intrinsic pathway activator(e.g., kaolin, celite, glass, ellagic acid, micronized silica, Hagemanfactor, etc.) or a combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes an extrinsic pathway activator(e.g., tissue factor, recombinant tissue factor, thromboplastin, etc.)or a combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes a coagulation activator (e.g.,thrombin, factor Xa, reptilase, ecarin, Russell's viper venom or othersnake venoms, etc.) or a combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes a platelet activator orplatelet inhibitor (e.g., GPIIb/IIIa inhibitors (e.g., abciximab,eptifibatide, tirofiban, roxifiban, orbofiban), cytochalasin D,blebbistatin, PAR1 inhibitors, PAR4 inhibitors, glycoprotein IBinhibitors, TRAP, ADP, arachidonic acid, ADP inhibitors, non-steroidalanti-inflammatory drugs, platelet activating factor, ristocetin,epinephrine, etc.) or a combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes a fibrinolytic functionsactivator or inhibitor (e.g., tPA, uKA, streptokinase, TAFIa,plasmin/plasminogen, aprotinin, epsilon-aminocaproic acid, tranexamicacid, plasminogen activator inhibitor 1 (PAI1), α2-antiplasmin (α2-AP),or plasmin-antiplasmin complexes, carboxypeptidase inhibitor) or acombination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes FXIIIa inhibitors or acombination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuit includes thrombomodulin or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuit includes low molecular weight heparin ora combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes Hexadimethrine bromide(polybrene) or a combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes heparin or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes corn trypsin inhibitor or acombination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes adenosine or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes GPRP (Gly-Pro-Arg-Pro) or acombination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes calcium or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes fibronectin or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes collagen or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes an immuno-detection reagent ora combination therewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes heparinase I or a combinationtherewith.

In some embodiments, the reagent, or combination of reagents, located inthe one or more fluidic circuits includes endothelial cells or activatedendothelial cells.

In some embodiments, the measurement system is selected from the groupconsisting of a sonorheometry-based system, thromboelastography-basedsystem, a thromboelastometry-based system, an optical-based system, afluorescence-based system, a colorimetric-based system, anaggregometry-based system, a resonance-based system, and an electricalimpedance-based system.

In another aspect, a method is disclosed of mixing a sample with one ormore reagents in an apparatus (e.g., a cartridge) and testing the mixedsample for the assessment of hemostasis. The method includes receiving aplurality of metered samples of from a plurality of metering chambersthat received test fluid from a sample holding tube (e.g., via amechanical coupling that connects the apparatus to the sample holdingtube or via an opening to which sample from the sample holding tube isplaced); alternately and multiplicatively flowing each of the aliquotsuntil the aliquot is mixed with a reagent, or a combination of reagents,to form a mixed aliquot, wherein the at least one aliquot alternatelyand cyclically flowed i) in a first direction from the metering chamberthrough one or more reagent pocket, with the one or more reagentstherein (e.g., lyophilized reagent bead), and along a serpentine pathwayin communication with the metering chamber until at least a portion ofthe aliquot reaches a detection zone located in, or after, theserpentine pathway and ii) in a second direction from the detection zonereversed to the first direction through at least a portion of theserpentine pathway toward the metering chamber until a trigger event;and driving the mixed aliquot in a testing chamber in fluidiccommunication with the metering chamber, wherein the testing chamber isstructurally configured for interrogation by a measurement systemconfigured to determine properties (e.g., mechanical properties orviscoelastic properties) of the mixed aliquot, and wherein aninterrogation of the testing chamber is performed with the mixed aliquotlocated therein.

In some embodiments, the method includes receiving the fluid in a firstchamber configured to substantially adjust the temperature of the testsample toward body temperature or other desired temperatures, whereinthe metered sample received in the metering chamber is received from thefirst chamber.

In some embodiments, the test fluid is moved into the first chamber inresponse to an applied pressure that is applied by, or generated from,the measurement system.

In some embodiments, the method includes conditioning the test fluid inthe first chamber to, or substantially near, a desired temperature,wherein the test fluid is mixed with the one or more reagents followingexit from the first chamber.

In some embodiments, the method includes isolating (e.g., blocking via avalve) the test fluid in the metering chamber to prevent the test fluidfrom contacting the one or more reagents during the filling of themetering chamber.

In some embodiments, a second applied positive or negative pressure isapplied by, or generated from, the measurement system (e.g., applied ata second pressure port in communication with the) at a second port incommunication with the serpentine pathway so as to move the at least onealiquot in the second direction.

In some embodiments, the first applied positive or negative pressure isapplied by, or generated from, the measurement system in reversed so asto move the at least one aliquot in the second direction.

In some embodiments, the operation of receiving the mixed aliquot in thetesting chamber further comprises receiving a negative pressure via thethird pressure port, wherein the third pressure port is further in fluidcommunication with the testing chamber.

In some embodiments, the testing chamber is downstream of the serpentinepathway and the third pressure port is downstream of the testingchamber.

These and other features and advantages of the present invention willbecome more readily apparent to those skilled in the art uponconsideration of the following detailed description and accompanyingdrawings, which describe both the preferred and alternative embodimentsof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments and together with thedescription, serve to explain the principles of the methods and systems:

FIG. 1 shows a perspective view of an example biological sample input ofa cartridge for use in a disposable system, in accordance with anillustrative embodiment.

FIG. 2 shows a side cross-sectional view of the example biologicalsample input of FIG. 1 with a casing, in accordance with an illustrativeembodiment.

FIG. 3 shows a side cross-sectional view of the example biologicalsample input of FIG. 2 with a sample holding tube attached thereon, inaccordance with an illustrative embodiment.

FIG. 4 shows a detailed view of the example biological sample input ofFIG. 3 , in accordance with an illustrative embodiment.

FIGS. 5A and 5B each shows the biological fluid pathways of four testingcircuits (e.g., Hemostasis testing circuits) that are located on asample preparation plane, in accordance with an illustrative embodiment.FIG. 5B further shows the cartridge body of FIG. 5A further coupled to asample holding tube, in accordance with an illustrative embodiment.

FIGS. 6A and 6B show a front perspective view and a back perspectiveview of FIGS. 5A, 5B, and 8 with labels corresponding to the heatingchamber filling.

FIGS. 6C and 6D show a front perspective view and a back perspectiveview of FIGS. 5A, 5B, and 8 with labels corresponding to the samplechamber filling.

FIGS. 6E and 6F show a front perspective view and a back perspectiveview of FIGS. 5A, 5B, and 8 with labels corresponding to the samplemixing and test chamber filling.

FIG. 7 shows the back-side of the cartridge of FIG. 5A and includes aninterconnection plane that interfaces to the sample preparation plane,that collectively form the biological fluid pathways for the testingcircuits, in accordance with an illustrative embodiment.

FIG. 8 shows portions of the biological fluid pathways that are on theinterconnection plane of FIG. 7 , in accordance with an illustrativeembodiment.

FIG. 9 shows an example testing chamber section for use with the examplecartridge, in accordance with an illustrative embodiment.

FIG. 10 shows a cross-sectional view of an example testing chamber inthe example testing chamber section, in accordance with an illustrativeembodiment.

FIG. 11 shows a detailed cross-sectional view of the example testingchamber of FIG. 10 , in accordance with an illustrative embodiment.

FIG. 12 shows a cross-sectional view of the disposable systemoperatively coupled to a measurement system, in accordance with anillustrative embodiment.

FIG. 13 shows an example shear modulus versus time curve, in accordancewith an illustrative embodiment.

FIG. 14 shows an example of shear modulus curves obtained with anactivator of coagulation and with and without a fibrinolysis inhibitor.A differential comparison of these curves can provide information aboutthe fibrinolytic activity of the sample.

FIG. 15 shows potential embodiments of differential metrics that can bemeasured from shear modulus curves obtained with an activator ofcoagulation and with and without a fibrinolysis inhibitor.

FIG. 16 shows a photograph of an exemplary cartridge of FIGS. 1, 2, 3,4, 5A, 5B, 7, 8 , and 9 for use in a disposable system, in accordancewith an illustrative embodiment.

FIG. 17 shows a front view of the exemplary cartridge of FIGS. 1, 2, 3,4, 5A, 5B, 7, 8 , and 9 in accordance with an illustrative embodiment.

FIG. 18 shows a front view of the exemplary cartridge of FIGS. 1, 2, 3,4, 5A, 5B, 7, 8 , and 9, in accordance with an illustrative embodiments.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter withreference to specific embodiments of the invention. Indeed, theinvention can be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will satisfy applicablelegal requirements.

As used in the specification, and in the appended claims, the singularforms “a,” “an,” “the,” include plural referents unless the contextclearly dictates otherwise.

The term “comprising” and variations thereof as used herein are usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms.

As used throughout, by a “subject” is meant an individual. The subjectmay be a vertebrate, more specifically a mammal (e.g., a human, horse,pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pigor rodent), a fish, a bird or a reptile or an amphibian. The term doesnot denote a particular age or sex.

The apparatus described here includes a single-use cartridge apparatusconfigured to facilitate in vitro assessment of one or more hemostaticfunctions. Hemostatic function refers to a functional role of variousblood components such coagulation factors, fibrinogen, platelets,fibrinolytic factors, and components of the vasculature. The cartridgeapparatus and associated measurement system, in some embodiments, areconfigured to assess hemostatic function by measuring changes in atleast one mechanical property of the tested sample when such sample isexposed to one or more reagents. In some embodiments, the cartridgeapparatus and its test chambers are configured to facilitatemeasurements of viscoelastic properties, e.g., based on interrogationusing ultrasound pulses or ultrasonic energy. However, otherinterrogation systems may be used with a cartridge apparatus with thefeatures described herein. Examples of other interrogation systemsincludes, for example, but not limited to, systems that employ cup/pintechnologies (such as in the case of thromboelastography andthromboelastometry), oscillating piston to measure changes in mechanicalimpedance, optical sensing, fluorescence sensing, colorimetric sensing,aggregometry, resonance sensing, or electrical impedance sensing, amongothers.

A broad array of reagents can be utilized in the cartridge apparatus,including intrinsic pathway activators (without limitations kaolin,Hageman factor, celite, glass, ellagic acid, micronized silica etc),extrinsic pathway activators (without limitations tissue factor,recombinant tissue factor, thromboplastin, etc), other coagulationactivators (without limitations thrombin, factor Xa, reptilase, ecarin,Russell's viper venom or other snake venoms, etc), platelet activatorsor platelet inhibitors (without limitations GPIIb/IIIa inhibitors (suchas abciximab, eptifibatide, tirofiban, roxifiban, orbofiban),cytochalasin D, blebbistatin, PAR1 inhibitors, PAR4 inhibitors,glycoprotein IB inhibitors, TRAP, ADP, arachidonic acid, ADP inhibitors,non-steroidal anti-inflammatory drugs, etc.), fibrinolytic functionactivators or fibrinolytic function inhibitors (without limitations tPA,uKA, streptokinase, TAFIa, plasmin/plasminogen, aprotinin,epsilon-aminocaproic acid, tranexamic acid, plasminogen activatorinhibitor 1 (PAI1), α2-antiplasmin (α2-AP), or plasmin-antiplasmincomplexes, carboxypeptidase inhibitor, etc.), and others (FXIIIainhibitors, Hexadimethrine bromide (polybrene), heparinase (e.g.,heparinase I), ristocetin, heparin, low molecular weight heparin, corntrypsin inhibitor, adenosine, GPRP, calcium, fibronectin, collagen,epinephrine, immuno-detection reagents, direct thrombin inhibitors,factor Xa inhibitors, reagents aimed at reversing or eliminating theeffects of the new oral anticoagulants (such as the direct thrombininhibitors and the factor Xa inhibitors), thrombomodulin, etc.).Additional non-functional reagents could also be used to preserve thefunctionality of the other reagents (buffers and stabilizers forlyophilization or drying, dyes, etc.).

Reagents, in some embodiments, are placed and stored in chambers (e.g.,pockets located within a fluidic circuit) in the cartridge apparatus butin alternative embodiments reagents can be placed and stored in variouschambers or fluidic channels in the fluidic circuit of the cartridgeapparatus. A fluidic circuit generally refers to one or more fluidicpathways established between sample preparation and the one or more testchambers where samples are ultimately measured.

In some embodiments, reagents are placed and stored in the cartridgeapparatus in liquid forms or can be lyophilized in spheres (such as inthe case of the Lyopheres™ produced by BioLyph LLC), lyophilized infilms, lyophilized on the plastic surfaces, dried on the plasticsurfaces, or spray coated, etc., in order to improve shelf-lifestability. A person of ordinary skills in the art should recognize thatthese reagents are not fully inclusive and other reagents or reagentcombinations that are inhibitors or activators of one or more hemostaticfunctions could be used in this cartridge.

The cartridge apparatus disclosed here is a component of a measurementsystem (e.g., a hemostasis measurement system). The measurement system(also referred to as the instrument) includes at least an interfaceelement which couples between the cartridge apparatus and a measuringelement configured to measures viscoelastic properties or mechanicalproperties of a sample processed within the cartridge apparatus. Themeasured viscoelastic properties or mechanical properties are outputtedas results to a user interface. An example user interface is describedin commonly assigned U.S. Pub. No. 2011/0252352 to Viola et al., whichis incorporated by reference herein in its entirety.

In some embodiments, the interface element includes one or more heatingand/or cooling elements.

In some embodiments, the interface element includes a fluidic manifoldthat facilitate connection to one or more pump elements and one or morevalves.

In some embodiments, the interface element includes one or more sensors,e.g., configured to perform hemostasis measurements. The one or moresensors, in some embodiments, includes ultrasound sensors. In otherembodiments, the one or more sensors includes other interrogativedevices that is based on thromboelastography, thromboelastometry (e.g.,a thromboelastography-based system or a thromboelastometry-basedsystem), or that measures changes in mechanical impedance, changes inperturbation as observed via an optical-based system (e.g., having anoptical sensor), fluorescence, colorimetric-based system,aggregometry-based system (e.g., having optical sensor, acoustic sensor,or electrodes that measure aggregation with the test sample),resonance-based system (e.g., having optical, acoustic, or mechanicalposition sensors that measures the sample when the sample is at, or nearresonance), electrical impedance-based system (e.g., having electrodesconfigured to measure electrical impedance), or a combination thereof.

In some embodiments, the interface element includes a mechanical clampconfigured to position the cartridge apparatus in a desired orientationwith respect to the components (the one or more sensors, the fluidicmanifold, the heating and/or cooling elements, and etc.) of themeasurement system. When the interface element is interfaced with thecomponents of the measurement system, the cartridge apparatus, in someembodiments, is driven via a series of controlled actions orchestratedby the measurement system to prepare the test sample for measurement. Insome embodiments, the preparation operations include sample aspirationof a sample from a sample container (also referred to as a sampleholding tube), sample heating and/or cooling, sample metering, samplemixing with reagents, and sample measurement. Each step, with referenceto various embodiments, is described below. After measurements arecompleted, the results are output in the instrument user interface.

In some embodiments, the cartridge apparatus and its internal componentsare the only component that directly contact with a sample to beanalyzed.

In some embodiments, the cartridge includes computer readableinformation that can be optically or communicatively interrogated (e.g.,RFID tags, computer readable medium such as flash ICs, QR codes, BARcodes, and etc.) and/or human readable information (e.g., labels).

The various embodiments described below does not utilize any activevalve element in the cartridge design, but instead relies on a fluidicmanifold and one or more valves placed in the instrument. Fluid is movedthrough the various cartridge components via pressure differentialand/or gravity and/or material properties (such as in the case ofhydrophobicity or hydrophilicity) and/or capillary forces.

In these embodiments, the cartridge is configured to couple with theinstrument via one or more connection ports that are aligned viaalignment slots. The connection ports include one or more pressure portsand one or more vent ports. However, in alternative embodiments,actuated valves (such as in the case of elastomeric valves) can beincluded in the cartridge design to control fluid flow. These valves areactuated, in some embodiments, by corresponding hardware and softwarecomponents in the measurement system.

The surface properties and texture of the cartridge surfaces in directcontact with the sample can be optimized to promote sample adhesionand/or sample flow. In some embodiments, the test chamber's interiorsurface and/or other interior surfaces of the fluidic circuit within thecartridge apparatus are plasma treated to optimize the surface energyand texture for adhesion of specific plasma proteins. In otherembodiments, test chamber's interior surface and/or other interiorsurfaces of the fluidic circuit are treated with surface roughnesstexturing, material coating (such as in the case of gold plating),biological material coating (such as in the case of fibronectin orcollagen coating, for example), raw material selection (e.g., use ofspecific plastic or other materials for the plate that does not requireadditional treatment), etc. Such treatments may be performedindependently, or in conjunction with, a plasma treatment. Similarly,the cartridge materials can be selected or manipulated to achieve thedesired hydrophobicity or hydrophilicity. These properties can bechanged by plasma treatment or by surface coatings.

As described in more details below, the cartridge and the associatedmeasurement system can utilize one or more sensors of one or more types(e.g., optics, pressure, ultrasound, etc.) as part of the automatedoperations of the cartridge. In addition, the outputs of such one ormore sensor(s) can be further utilized to perform quality controlchecks. These checks may be performed before, during, or after cartridgetesting to ensure function of one or more of the subsystems (forexample, ultrasound or other interrogation system, fluidics, fluidlevel, clamping, cartridge positioning/orientation system, ortemperature control), ensure the cartridge is functional, ensure correctsample preparation before measurements are performed or have beenperformed for the measurement, and may also be used to accept or rejecta test result or even to abort testing before initiation ofmeasurements.

Note that in the discussion below a fluidic circuit includes a channelwith fluidic component that connects one or more chambers together.Fluid circuit is also referred to as a testing channel in a multitude ofchannels that can be individually and controllably processed within asingle cartridge apparatus.

Cartridge Input Section

FIGS. 1, 2, 3, and 4 are schematic illustrations of an examplebiological sample input section of a cartridge 100 for evaluatinghemostasis. Specifically, FIG. 1 shows a perspective view of an examplebiological sample input of a cartridge for use in disposable system, inaccordance with an illustrative embodiment. FIG. 2 shows a side view ofthe example biological sample input of FIG. 1 with a casing, inaccordance with an illustrative embodiment. FIG. 3 shows a sidecross-sectional view of the example biological sample input of FIG. 2with a sample holding tube attached thereon, in accordance with anillustrative embodiment. FIG. 4 shows a detailed view of the examplebiological sample input of FIG. 3 , in accordance with an illustrativeembodiment. In alternative embodiments, the input section of thecartridge comprises a well to which fluid sample can be placed, forexample, by way of a pipette or tube.

In some embodiments, and as shown in FIG. 1 , the cartridge 100 has adual connection tab 28 a, 28 b for coupling the cartridge 100 to asample container guide 1 (shown in FIG. 2 ). As shown in FIG. 2 , thesample container guide 1, when mated with the cartridge 100, aligns asample container 2 to a sample input port 3 of the cartridge 100. Thecartridge 100 also includes an alignment tab 29 that is configured toslide into an alignment groove 30 of the sample container guide 1 tofurther stabilize the coupling of the sample container guide 1 to thecartridge 100. The sample container guide 1 can further provide a hardstop 5 (shown in FIG. 3 ) to hold the sample container 2 at theappropriate height to establish fluidic communication with the cartridge100.

In various embodiments, the sample container 2 is an evacuated tube(also referred to herein as the sample holding tube 2) such as a BDVacutainer™ tube, and the sample input port 3 comprises one or moreneedles required for sample transferring 3 a and venting 4 (see FIG. 1). Though shown as concentric in the figures, the needles can beconfigured to be concentric, side by side, or integrated. In someembodiments, and as shown in FIG. 1 , the sample transferring needle 3 aincludes inlets (3 b and 3 d) and an outlet 3 c that terminates in asample inlet chamber 26 of the cartridge 100. The sample inlet chamber26 is in fluid communication with an inlet pathway 8 that leads to aretention/heating chamber 6 (see FIG. 5A). In some embodiments, and asshown in FIG. 1 , the venting needle 4 includes an outlet 4 a that isconfigured to terminate within the sample container 2 when attached andis spaced apart from the inlet 3 d so as to minimize bubbles being drawninto the inlets 3 b and 3 d. The venting needle 4 also has an inlet 4 bthat terminates in a venting inlet chamber 27 of the cartridge. Theventing inlet chamber 27 is in fluid communication with a vent pathway 9that, in some embodiments, terminates at a filter chamber 9 a (shown inFIG. 5A) housing a filter. An alternative sample container 2 can beutilized, such as a syringe, which requires a luer lock connection onthe cartridge 100. And, as noted above, in other embodiments, the inputsection of the cartridge comprises a well to which fluid sample can beplaced, for example, by way of a pipette or tube.

Vent Pathway

FIGS. 5A, 5B, 7, and 8 are schematic illustrations of biological fluidpathways of the example cartridge 100 in accordance to an embodiment.Specifically, FIGS. 5A and 5B each shows the biological fluid pathwaysof four testing circuits (corresponding to test chambers 16 a, 16 b, 16c, and 16 d, shown in FIG. 5A), also referred to herein as Hemostasistesting circuits, that are located on a sample preparation plane, inaccordance with an illustrative embodiment. Though shown with fourtesting circuits, additional circuits or less may be included,including, e.g., two, three, five, six, seven, eight, and etc. FIG. 5Bfurther shows of the cartridge body of FIG. 5A further coupled to asample holding tube 2, in accordance with an illustrative embodiment.FIG. 7 shows the back-side of the cartridge of FIG. 5A and includes aninterconnection plane that interfaces to the sample preparation plane,that collectively form the biological fluid pathways for the testingcircuits, in accordance with an illustrative embodiment. FIG. 8 showsportions of the biological fluid pathways that are on theinterconnection plane of FIG. 7 , in accordance with an illustrativeembodiment.

As noted above, the biological fluid pathways are formed on, and across,multiple planes defined in the cartridge 100. A first plane of fluidpathways of the cartridge 100 is shown in FIGS. 5A and 5B. The firstplane of fluid pathways of the cartridge 100 may alternatively bereferred to as a front plane of the cartridge 100. FIGS. 7 and 8 eachshows a second plane of fluid pathways of the cartridge 100 in whichFIG. 8 shows the fluid pathways in the second plane isolated from theremainder of the structure of the cartridge 100 for ease ofunderstanding. The second plane of fluid pathways of the cartridge 100may alternatively be referred to as a back plane of the cartridge 100.The fluid pathways between the first and second planes are connected byfluidic vias that traverse across the various planes of the cartridge100.

As discussed above in relation to FIG. 1 , in some embodiments, theventing inlet chamber 27 is in fluid communication with a vent pathway9. The vent pathway 9 may terminate at a filter chamber 9 a (shown inFIG. 5A), which may house a filter therein. The filter chamber 9 a inthe first plane of fluid pathways of the cartridge 100 is in fluidcommunication with a vent port 22 i (shown in FIG. 8 ) in the secondplane of fluid pathways of the cartridge 100. As discussed in moredetail below, cartridge 100 couples with the measurement system (alsoreferred to herein as the instrument) via the vent port 22 i to provideatmospheric pressure to vent pathway 9.

Heating Chamber Pathway

As discussed above in relation to FIG. 1 , in some embodiments, thesample transferring needle outlet 3 c terminates in a sample inletchamber 26 of the cartridge 100. The sample inlet chamber 26 is in fluidcommunication with an inlet pathway 8. The sample inlet pathway 8provides a fluid communication pathway between the sample inlet chamber26 and a retaining/heating chamber 6 (also referred to herein as heatingchamber 6 or as a “first chamber”) (shown in FIG. 5A). The label“first”, “second”, and “third” as used herein is provided merely aslabels and do not intended to connote a sequence. The heating chamber 6is configured to mate with a corresponding thermal regulating (e.g.,heating/cooling) system in the measurement system to warm or cool thesample toward or to, or near, a pre-defined temperature.

The heating chamber 6, as provided herein, facilitate uniformconditioning of the test fluid prior to the fluid be metered oraliquoted to their respective testing, thus reducing variability in thetest sample that can affect subsequent measurements and analysis. Theshape of the heating chamber 6 can be optimized for heating/coolingtransfer, as in the case here in which a thin cross-section with thinwalls is used. The materials of the cartridge 100 can also be optimizedto facilitate heating/cooling. In some embodiments, the sampleheating/cooling conditioning stage can also be implemented in one ormore chamber/channels of the cartridge design and it is not limited tojust occur within just the heating chamber 6. In some embodiments, astirring, rotating, or oscillating element (not shown) can be placed inthe heating chamber 6 that may be controlled by the measurement systemto promote uniform temperature heating or cooling. In other embodiments,test fluid in the heating chamber 6 may be vibrated by the measurementsystem vibrating the cartridge 100 to promote uniform temperatureconditioning of the test fluid.

In some embodiments, temperature measurement is conducted of the testsample in the cartridge 100. To measure the temperature, a sensor can beincorporated in the measurement system or in the cartridge 100. In someembodiments, a thermistor or thermocouple can be placed in physicalcontact with the cartridge 100, or biological sample (such as blood). Inother embodiments, an IR thermometer is pointed at the cartridge 100 orbiological sample. In either case the cartridge 100 may incorporate asmall well through which the incoming blood passes, rather than havingdirect contact with the blood. In some embodiments, the temperature ofthe test sample may be assessed at or near the heating chamber 6. Inother embodiments, the temperature of the test sample may be assessedwhile the test sample is flowing through channels as it is directedtoward the test chambers 16.

Referring now to FIGS. 5A, 5B, and 8 , the sample inlet pathway 8terminates at a first corner 6 a of the heating chamber 6, shown as thetop left corner of heating chamber 6 in FIGS. 5A and 5B. In someembodiments, chambers along the fluid pathways are generally filled fromthe top as to prevent blood to backflow into the inlet. A fill outletchannel 10 a extends from a second corner 6 b of the heating chamber 6opposite from the first corner 6 a.

The fill outlet channel 10 a extends to a filter chamber 10 with afilter therein. The filter chamber 10 (e.g., as shown in FIGS. 5A and5B) in the first plane of fluid pathways of the cartridge 100 is influid communication with a heating-chamber fill channel 10 b shown inthe second plane of fluid pathways of the cartridge 100 (see FIG. 8 ).The fill conduit 10 b is in fluid communication with a pressure port 22a (see also FIG. 8 ) that facilitate filling of the heating chamber 6.The conduit 10 b is a part of a network of conduits used to consolidatepressure ports (e.g., 22 a as discussed, as well as 22 b-22 i to belater discussed) of the cartridge 100 to one or more areas to which themeasurement system can coupled with its pressure control interfaces.Such configuration reduces the complexity of the measurement system tocontrol fluid movement within the cartridge 100. Indeed, the conduitsthat handles the control of movement of the fluid sample in the firstplane of the cartridge 100 is primarily placed in the second plane ofthe cartridge 100. FIGS. 6A and 6B show a front perspective view and aback perspective view of FIGS. 5A, 5B, and 8 with additional labelscorresponding to the description of this section.

Heating Chamber Fill

In operation, the instrument's fluid pump aspirates the sample throughthe input port 3 (see FIGS. 1-2 ) via the connection ports 22 (see FIG.7 ) (also referred to herein as pressure ports) and into the heatingchamber 6 (see FIG. 5A or 5B) of the cartridge 100. For example, theinstrument's fluid pump may be in communication with and applydifferential pressure (e.g., positive or negative) to a pressure port 22a (see FIG. 8 ). This in turn creates an applied pressure along the fillconduit 10 b, within the heating chamber 6, and along the inlet pathway8 to aspirate the sample into the heating chamber 6. At the same time,the inner vent needle 4 is linked to the isolated pathway 9 thatreceives atmospheric pressure from the instrument via the vent port 22 i(see FIG. 8 ) to neutralize pressure in the sample container 2 as thesample is aspirated into the heating chamber 6 of the cartridge 100.During filling of the heating chamber 6, all other ports (e.g., 22b-221) are closed, e.g., by the measurement system.

When the heating chamber 6 is filled, the filter within filter chamber10 is clogged and creates a pressure spike that is detected by theinstrument, causing the instrument to turn off the fluidic pump. Theinstrument may also close the vent port 22 i or otherwise discontinuesupplying atmospheric pressure via vent port 22 i upon detecting thepressure spike. Alternative filling detection techniques could also beused, i.e., optical sensors placed at the desired fill level, volumetriccontrol, fixed time of pressure alteration (negative and/or positivepressures), ultrasound detectors placed at the desired fill level, etc.The sample remains in the heating chamber until the desired temperatureis reached, which can for example be at or near body temperature of anormal and typical subject (e.g., about 37° C. for a healthy person). Inother instances, other desired temperatures may be warranted. The shapeof the heating chamber 6 and the channels leading to the sample meteringchambers 11 (described below) are configured so that bubbles that mightbe present in the fluid sample are trapped away from the rest of thefluidic circuit. The shape of the inlet pathway 8 includes ananti-siphon feature 8 a (see FIGS. 5A and 5B) and is configured toreduce the occurrence of bubbles forming in the heating chamber 6 andprevent siphoning to and from the sample container 2. To this end,additional unprocessed test sample (e.g., un-warmed blood) is preventedfrom being siphoned into the heating chamber following the first drawntest sample being heated and/or cooled, e.g., when the processed testsample is pulled into the metering chamber 11 (also referred to hereinas sample chamber 11 and “second” chamber). FIGS. 6A and 6B also showlabels corresponding to the description of this section.

Sample Aliquot (Metering) Chambers Pathway

Referring to FIGS. 5A, 5B, and 8 , a first side 6 c of the heatingchamber 6 (see FIG. 5A) extends between the first corner 6 a and thesecond corner 6 b.

One or more of outlet ports 6 e-6 h (see FIG. 5A) are arranged along thelength of the second side 6 d of the heating chamber. Each of the one ormore outlet ports 6 e-6 h may be arranged in a different one of thevalleys along the second side 6 d of the heating chamber in which thevalleys direct the sample into conduits leading to each respective testchannel. A test channel, in some embodiments, refers to the associatedfluidic pathway structures and testing chamber, collectively, used toperform a measurement for a given aliquot sample. In some embodiments,and as shown in FIG. 5B, each of the one or more outlet ports 6 e-6 h inthe first plane of fluid pathways of the cartridge 100 is in fluidcommunication with a first end of a corresponding one or more channels20 a-20 d (see FIG. 8 ) in the second plane of fluid pathways of thecartridge 100, collectively referred to as channels 20 (see FIG. 5B). Asecond end of each of the one or more channels 20 a-20 d (see FIG. 8 )in the second plane of fluid pathways of the cartridge 100 is likewisein fluid communication with a first end of a corresponding one or morechannels 11 a-11 d (see FIG. 5B) in in the first plane of fluid pathwaysof the cartridge 100. A second end of each of the one or more channels11 a-11 d terminates in a corresponding one or more sample chambers 11(shown in duplicates (“×4”) in FIG. 5B with an “o” symbol therein). Inthe example shown in FIGS. 5A, 5B, 7, and 8 , there are four samplechambers 11. More or fewer sample chambers 11 and corresponding fluidcommunication pathways with the heating chamber 6 may be present on thecartridge 100 in some configurations.

The sample chambers 11 are fed by the one or more channels 20originating from the bottom of the heating chamber 6. This geometricconfiguration avoids bubbles being drawn into the sample chambers 11 asthe bubbles rise to the upper portion of the heating chamber 6.

Each of the sample chambers 11 has a corresponding fill channel 11 ethat is in fluid communication with a corresponding filter chamber 12(shown in duplicates (“×4”) in FIG. 5B with a “+” symbol) with a filtertherein. The filter chamber 12 in the first plane of fluid pathways ofthe cartridge 100 is in fluid communication with a channel 12 a (seeFIG. 8 ) in the second plane of fluid pathways of the cartridge 100. Thechannel 12 a is in fluid communication with a pressure port 22 g (seeFIG. 8 ).

In some configurations, when more than one sample chamber 11 areimplemented on the cartridge 100, the channel 12 a (see FIG. 8 ) is influid communication with all of the sample chambers 11 (see FIG. 5B) viacorresponding fill channels 11 e and filter chambers 12. Accordingly,channel 12 a acts as a manifold for applying negative pressure to all ofthe sample chambers 11 via a single pressure port 22 g. Therefore, aseparate pressure port is beneficially not needed for filling each ofthe sample chambers 11. FIGS. 6C and 6D show a front perspective viewand a back perspective view of FIGS. 5A, 5B, and 8 with additionallabels corresponding to the description of this section.

Heating Chamber Vent Pathway

Referring to FIGS. 5A, 5B, and 8 , the heating chamber 6 comprises avent pathway 31 a, 31 b, 31 c along the first side 6 c of the heatingchamber for venting the heating chamber 6 as the sample chambers 11 arefilled. The vent pathway 31 (not shown) includes the fluid pathwaythrough conduit elements 31 a-31 d. Channels 31 a-31 b terminate at oneend in the heating chamber 6 along the first side 6 c and terminate atthe other end at a filter chamber 31 c with a filter therein.Accordingly, channels 31 a-b provide a fluid pathway between the heatingchamber 6 and the filter chamber 31 c. The filter chamber 31 c (see FIG.5A) in the first plane of fluid pathways of the cartridge 100 is influid communication with a channel 31 d (see FIG. 8 ) in the secondplane of fluid pathways of the cartridge 100, which in turn is in fluidcommunication with a vent port 22 c (see FIG. 8 ). As discussed in moredetail below, cartridge 100 couples with the instrument via the ventport 22 c for the instrument to provide atmospheric pressure to vent theheating chamber 6. FIGS. 6C and 6D also show labels corresponding to thedescription of this section.

Sample Chamber Vent Pathway

Referring to FIGS. 5A, 5B, and 8 each of the sample chambers 11 includesa vent pathway 18 that terminates at a first end in a correspondingsample chamber 11. A second end of the vent pathway 18 in the firstplane of fluid pathways of the cartridge 100 is in fluid communicationwith a vent manifold 18 in the second plan of fluid pathways of thecartridge 100. When more than one sample chamber 11 is present, all ofthe vent pathways 18 (see FIG. 5B) of the sample chambers are in fluidcommunication with the vent manifold 18 a (see FIG. 8 ). The ventmanifold 18 a (see FIG. 8 ) in the second plane of fluid pathways of thecartridge 100 is further in fluid communication with a first end of achannel 18 b (see FIG. 5B) in the first plane of fluid pathways. Asecond end of channel 18 b (see FIG. 5B) is in fluid communication witha filter chamber 18 c (see FIG. 5B) with a filter therein. Filterchamber 18 c (see FIG. 5B) in the first plane of fluid pathways of thecartridge 100 is in fluid communication with a vent port 22 e (see FIG.8 ). FIGS. 6C and 6D also show labels corresponding to the descriptionof this section.

Sample Aliquot (Metering) Chambers Fill

During operation, once the sample is at, or near, the desiredtemperature, the sample is aliquoted (or metered) into one or moreindependent sample chambers 11 (see FIG. 5B). Referring to FIG. 5Bunless indicated otherwise, in various embodiments, the sample chambersare filled by applying a negative pressure at pressure port 22 g (seeFIG. 8 ) via a pump in the instrument while venting the heating chamber6 by way of the vent port 22 c (see FIG. 8 ). Each filter chamber 12 hasa filter therein that will clog when the corresponding sample chamber 11is filled and will trigger the pressure sensor of the instrument to turnthe pump off, similar to filling the heating chamber 6. As discussedabove, alternative filling detection techniques could be used. Invarious embodiments, all the sample chambers 11 are controlled by asingle valve and fluidic pathway in the instrument via pressure port 22g (see FIG. 8 ). The cutoff pressure will not trigger until all samplechambers' filters clog in the corresponding filter chambers 12. Thesample chambers 11 are used to separate samples into independentfunctional channels, aliquot a known volume of sample, and stage thesample for mixing with reagents. While the sample chambers 11 arefilled, pressure ports 22 b, 22 d, 22 f, and 22 h (see FIG. 8 ) as wellas vent port 22 e (see FIG. 8 ) are closed by the instrument so as toprevent fluid from leaking past the location 19, arranged below thesample chambers 11. Once the sample chambers 11 have been filled, thevent port 22 e (see FIG. 8 ) is opened to atmospheric pressure so thatthe one or more sample chambers 11 can be fluidically separated fromeach other and the heating chamber 6. Vent port 22 e (see FIG. 8 )remains open to atmosphere during sample mixing as discussed below.FIGS. 6C and 6D also show labels corresponding to the description ofthis section.

Mixing and Testing Pathway

Referring to FIG. 5B unless indicated otherwise, each of the samplechambers 11 is in fluid communication with a corresponding one or morereagent pocket 14 (see FIG. 5A) configured to house at least onelyophilized bead comprising a reagent. As shown in FIGS. 5A and 5B, tworeagent pockets 14 (shown as 14 a and 14 b in FIG. 5A for one of thetest channels) are provided. In other embodiments, a single reagentpocket is used for each testing channel. In yet other embodiments, morethan two reagent pockets 14 are used for each testing channel. Thereagent pockets 14 are in turn in fluid communication with a serpentinechannel 13 (see FIG. 5A, and shown with a duplication symbol (“×4”).Each of the serpentine channel 13 has a first end in fluid communicationwith the reagent pockets and a second end that terminates at an opticaldetection zone 15. As discussed below, the instrument (i.e., measurementsystem) may optically interrogate the optical detection zone 15 of thecartridge 100 to facilitate the control of a pump that facilitatesmixing of the individual aliquots with the corresponding reagent(s).Each of the serpentine channel 13 (see FIG. 5A) is in fluidcommunication with a test chamber 16 (see FIG. 5B, with duplicationsymbol “×4”) which in turn is in fluid communication with a filterchamber 17, with a filter therein. The filter chamber 17 in the firstplane of fluid pathways of the cartridge 100 is in fluid communicationwith a first end of a corresponding one of fluid channels 17 a-17 d (seeFIG. 8 ) in the second plane of fluid pathways of the cartridge 100. Asecond end of each of the fluid communication channels 17 a-17 d (seeFIG. 8 ) are in fluid communication with a corresponding pressure port22 b, 22 d, 22 f, and 22 h (see FIG. 8 ). The cartridge 100 couples withinstrument via the pressure ports 22 b, 22 d, 22 f, and 22 h (see FIG. 8) to supply positive and negative pressure to facilitate mixing andtesting of the sample as described below. FIGS. 6E and 6F also show afront perspective view and a back perspective view of FIGS. 5A, 5B, and8 with additional labels corresponding to the description of thissection.

Sample Mixing

Referring to FIG. 5B unless indicated otherwise, each individual aliquotin the sample chambers 11 is pulled into a separate reservoir or channel(a serpentine channel pathway 13 (see FIG. 5A) in various embodiments)and brought into contact with the channel specific reagent, located inone (or both) of the two reagent pockets 14 (see FIG. 5A). Specifically,a pump in the instrument applies negative pressure to pressure ports 22b, 22 d, 22 f, and 22 h (see FIG. 8 ) to draw the sample through thereagent pockets 14 (see FIG. 5A) and serpentine channel pathway 13 (seeFIG. 5A). The reagents and sample are kept separate from each otherduring sample chamber 11 filling so as to avoid reagents floating on theblood and getting trapped in the filter in filter chambers 12, as wellas to ensure fluidic isolation of the one or more sample chamber 11 fromthe heating chamber 6. To this end, precise time of when the test sampleis in contact with the reagents can be measured thereby facilitatingaccurate and precise clot time measurements (e.g., from when mixingbegins). In addition, the reagents and test samples are kept separatedfrom one another, in some embodiments, until all channels are metered toprevent undesired siphoning of test samples from other channels or fromthe unprocessed sample in the fluidic pathways. The sample is aspiratedthrough the serpentine channel 13 (see FIG. 5A) until it triggers anoptical sensor in the instrument (detection zone is top of serpentinechannel near the optical detection zone 15 (see FIG. 5A)) which closesoff the channel from the pump. Mixing in the serpentine channels orzones, or region, therein can be controlled by one or more independentvalves and pathways that allow individual channel control. Alternativesensor techniques could be utilized: pressure, pass through opticalsensors, ultrasound detection, time, volumetric control, etc. Once allthe channels' optical sensors trigger, the pump reverses and appliespositive pressure to pressure ports 22 b, 22 d, 22 f, and 22 h (see FIG.8 ). The positive pressure pushes the biological sample, such as blood,down the serpentine path 13 (see FIG. 5A) for a designated time, oruntil a second set of optical sensors in the instrument is tripped (inan alternative embodiment). This process, to pull the sample up theserpentine path 13 (see FIG. 5A) to the optical detection zone where itis detected by an optical sensor of the instrument and push back for agiven time, repeats until full sample mixing is achieved. FIGS. 6E and6F also show labels corresponding to the description of this section.

Other sensors (e.g., impedance sensors), pressure sensor, and etc., maybe used. Alternatively, additional sensors may be used to detect bothends of the optical detection zone. Alternate pathway geometries,obstructions to create turbulence, cycle numbers, and cycle speed areall design alternatives that can be used with varying test types toachieve optimal results. In alternative embodiments, mixing could beachieved with one or more ferromagnetic beads or bars placed within thecartridge and controlled by the instrument.

Test Chamber Filling

Referring to FIGS. 5A, 5B, and 8 , one or more testing chambers 16 arefilled after mixing is completed. Using one or more independent valvesand pathways, each testing chamber 16 is filled with a sample via theapplication of pressure perturbations (negative and/or positivepressure) at pressure ports 22 b, 22 d, 22 f, and 22 h. Specifically,the instrument pump will apply negative pressure to pressure ports 22 b,22 d, 22 f, and 22 h until all of the filters are clogged in the one ormore filter chambers and cause a pressure spike to cause the instrumentto turn the pump off, similar to filling the heating chamber 6. Asdiscussed above, alternative filling detection techniques could be used.The testing chambers 16 have design features such as the ridges 24 awhich prevent the formation of bubbles in the testing chambers 16 duringfilling. Once filled, the instrument begins viscoelastic testing of thesample. FIGS. 6E and 6F also show labels corresponding to thedescription of this section.

In some embodiments, the cartridge apparatus includes, at least, fourindependent fluidic circuits configured with different sets of reagentsfor measurements (and/or sample preparation) to be performed inparallel. The measurements are performed per channel of the, at least,four channels of the cartridge. The measurement, in some embodiments,include viscoelastic properties such as a sample shear modulus. Themeasurement, in another embodiment, includes other properties such asviscosity, elastic modulus, or any other mechanical property of thesample, or combinations thereof.

Table 1 provides an example set of reagents and measurement parametersfor use in an example cartridge apparatus (e.g., apparatus 100, amongothers). As shown in Table 1, Channel #1 in the example cartridgeapparatus is interrogated to measure clot time of the test sample in thepresence of kaolin, which is an activator of the intrinsic pathway ofcoagulation. As shown in Table 1, Channel #2 is interrogated to measureclot time of the test sample in the presence of kaolin and in furtherpresence of heparinase I, which is a neutralizer of the anticoagulantheparin. As shown in Table 1, Channel #3 is interrogated to measureoverall clot stiffness of the test sample in the presence of i)thromboplastin, which is an activator of the extrinsic pathway ofcoagulation, and ii) polybrene, which is a neutralizer of theanticoagulant heparin. As shown in Table 1, Channel #4 is interrogatedto measure clot stiffness of the test sample with the same reagents aschannel #3, but with the addition of abciximab (e.g., Clotinab® and/orReoPro®), which is an inhibitor of platelet aggregation/contraction. Asshown in Table 1, when the assay is configured to operate with citratedwhole blood samples, calcium is added to all the reagent formulations.

TABLE 1 Reagents utilized in a preferred embodiment Channel # ReagentsMeasurement (units) 1 Kaolin, calcium, buffers Clot time and stabilizers(Seconds) 2 Kaolin, heparinase I, calcium, Clot time buffers andstabilizers (Seconds) 3 Thromboplastin, polybrene, Clot stiffnesscalcium, buffers and stabilizers (hecto Pascals) 4 Thromboplastin,polybrene, Clot stiffness abciximab (and/or (hecto Pascals) cytochalasinD), calcium, buffers and stabilizers

Table 2 provides an additional example set of reagents and measurementsfor use in an example cartridge apparatus (e.g., apparatus 100, amongothers). As shown in Table 2, channel #2 includes an extrinsic pathwayactivator with inhibition of fibrinolysis by tranexamic acid (TXA). Inaddition to the measurements previously presented in Table 1, channels#2, channel #3, and channel #4 are interrogated to also measure clotstiffness changes, which, for example, can be related to thefibrinolytic process. In some embodiments, other channels can includereagents that inhibit fibrinolysis and can also be interrogated tomeasure clot stiffness changes. For example, channel #4 could alsoinclude TXA or other fibrinolysis inhibitor in order to measure clotstiffness in the absence of fibrinolysis.

TABLE 2 Reagents utilized in a preferred embodiment Channel # ReagentsMeasurement (units) 1 Kaolin, calcium, buffers Clot time (Seconds) andstabilizers 2 Thromboplastin, polybrene, Clot stiffness calcium,tranexamic (hectoPascals) and acid, buffers and stabilizers Clotstiffness change 3 Thromboplastin, polybrene, Clot stiffness (hectocalcium, buffers and stabilizers Pascals) and clot stiffness change 4Thromboplastin, polybrene, Clot stiffness abciximab (and/or (hectocytochalasin D), calcium, Pascals) and clot buffers and stabilizersstiffness change

In some embodiments, clot time and clot stiffness are measured byanalyzing a shear modulus (clot stiffness) versus time curve that isgenerated within each measurement channel in the cartridge. FIG. 13shows an example shear module versus time curve, in accordance with anillustrative embodiment. Clot time may be determined by identifying whenthe clot stiffness meets or exceeds a threshold value, or when the firstor higher derivative of such property being measured meets or exceeds athreshold value, or at the point of maximum acceleration in the rate ofclot stiffness, or some combination of the above methods. Clot stiffnessmay be estimated by the clot stiffness at a fixed time after clot time,or the maximum overall clot stiffness measured within some time limit,or the clot stiffness at the point of maximum rate of change in clotstiffness, or some combination of the above methods. Similar methods canalso be applied to measure the effects of fibrinolysis (i.e., clotdissolution) and the corresponding reduction in clot stiffness. In someembodiments, clot stiffness changes can be calculated as percentage dropin clot stiffness over a fixed time window, as a rate of change of clotstiffness over time, as area under or over the clot stiffness vs timecurve within a predefined time window, as the time required to achieve apredefined drop in clot stiffness, or combinations thereof. Similarcurves and similar measurements to those just described can be formed byplotting the Young's modulus, viscosity, or other viscoelastic propertyof the sample being measured.

TABLE 3 Parameters reported from measurement of the preferredembodiments discussed in relation to Table 1. Hemostatic Index UnitsDescription Measurement Clot Time Minutes Clot time Clot time measured(min) in citrated from channel #1 whole blood with kaolin activation(intrinsic pathway) Heparinase Minutes Clot time Clot time measured ClotTime (min) in citrated from channel #2 whole blood with kaolinactivation with heparin and heparinase I neutralization Clot hectoStiffness of Clot stiffness Stiffness Pascals the whole measured from(hPa) blood clot channel #3 with thromboplastin activation (extrinsicpathway) and polybrene Fibrinogen hecto Contribution of Clot stiffnessmeasured from Contribution Pascals functional channel#4 withthromboplastin (hPa) fibrinogen activation, polybrene, and to clotstiffness abciximab Clot Time Unit Assessment of Calculated ratio ofRatio less residual heparin clot time values anticoagulation fromchannels #1 and #2 Platelet hecto Contribution of Calculated fromsubtraction Contribution Pascals platelet activity of the clot stiffnessvalues (hPa) to clot stiffness from channels #3 and #4

A person of ordinary skills in the art should recognize that clot timeand clot stiffness can be estimated using a number of methodologies andcriteria. Clot times and clot stiffness values obtained from the, atleast, four channels/measurements may be combined to provide, at least,six parameters can depict a functional status of the patient'shemostatic system. The indexes are summarized in Table 3. Relationshipbetween results (clot time, clot stiffness, clot stiffness change, etc.)from different channels may be verified to be within expected ranges asadditional quality control checks to verify instrument, cartridge, andsample function.

In other embodiments, other reagents can be used and other hemostaticindexes or output parameters can be obtained such as in the case of afibrinolytic index, indexes corresponding to the functionality ofanti-platelet treatments, indexes corresponding to the functionality ofanti-coagulation treatments, etc.

For example, one or more fibrinolysis indexes could be formed using theclot stiffness changes measured in any of the channels presented inTable 2, but preferably channels #3 and #4. Alternatively, afibrinolysis index could be formed by differential combination of theclot stiffness changes measured in channels #2 and channel #3 presentedin Table 2. Such combination could be in the form of a ratio, adifference, or combinations thereof. One of the benefit of using acombination of clot stiffness changes measured with and without ananti-fibrinoltyic reagent is the ability to mitigate the interferingeffects of non-fibrinolysis driven reductions in clot stiffness values.In some embodiments, TXA or other fibrinolysis inhibitor reagent can beincluded in both channel #2 and channel #4 of the example cartridge ofTable 2. With such modifications the parameters Clot Stiffness, PlateletContribution, and Fibrinogen Contribution could be derived without theinfluence of fibrinolysis by combination of the clot stiffnessmeasurements obtained in channel #2 and channel #4.

As discussed above, an example user interface is described in commonlyassigned U.S. Pub. No. 2011/0252352 to Viola et al., which isincorporated by reference herein in its entirety. The example userinterface may be used to display the measured hemostatic indexes asdiscussed in relation to Table 4, among other parameters.

TABLE 4 Parameters reported from measurement of the preferredembodiments discussed in relation to Table 2. Hemostatic Index UnitsDescription Measurement Clot Time Minutes Clot time in Clot timemeasured (min) citrated whole from channel #1 blood with kaolinactivation (intrinsic pathway) Clot hecto Stiffness of the Clotstiffness measured Stiffness Pascals whole blood from channel #3 with(hPa) clot thromboplastin activation (extrinsic pathway) and polybreneFibrinogen hecto Contribution of Clot stiffness measured ContributionPascals functional from channel #4 with (hPa) fibrinogen thromboplastinactivation, to clot polybrene, and abciximab stiffness Platelet hectoContribution of Calculated from subtraction Contribution Pascalsplatelet activity of the clot stiffness values (hPa) to clot stiffnessfrom channels #3 and #4 Clot % or Clot stiffness Changes (% or rate ofchange) Stiffness hPa/sec change over in clot stiffness measured Changeor sec time from channels #2 and #3 Clot % or Differential rateDifferential comparison Reduction hPa/sec, of clot stiffness of clotstiffness change Differential or no changes with measured in channelsunits and without #2 and #3. anti-fibrinolytic

As noted before, in various embodiments, the testing chambers 16 areshaped to facilitate ultrasound testing of viscoelastic properties ofthe sample, but alternative geometries can also be implemented tofacilitate other types of testing. Such an ultrasound testing system isdescribed in commonly assigned U.S. Pat. No. 9,726,647 and U.S. Pub. No.2016/0139159, both of which are hereby incorporated by reference intheir entirety. Ultrasound transducers in the measuring system connectwith the testing chambers 16 of the cartridge 100 via compliant anddeformable elastomers 21 which are affixed to a testing block 21 d onthe cartridge 100.

Example elastomeric materials optionally include, Dynaflex D3202,Versaflex OM 9-802CL, Maxelast 54740, RTP 6035, Versaflex CL2003X, amongothers. Referring now to FIG. 9 unless indicated otherwise, the testingblock 21 d is aligned with the testing chambers 16 (see FIG. 5B) viaalignment slots 23 and 24 on the cartridge 100. Referring still to FIG.9 , the elastomers 21 may be affixed to the testing block 21 d via aflange 21 a on the elastomers 21. The flange 21 a may have a pluralityof alignment holes 21 b that may receive corresponding alignment pegs(not shown) from the testing block 21 d. The soft elastomers 21 may alsoeach include a lens 21 c that focuses ultrasound energy within thesample at the testing chambers 16.

FIG. 16 shows a photograph of an exemplary cartridge of FIGS. 1, 2, 3,4, 5A, 5B, 7, 8 , and 9 for use in a disposable system, in accordancewith an illustrative embodiment. FIG. 17 shows a front view of theexemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9 , inaccordance with an illustrative embodiment. FIG. 18 shows a front viewof the exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9 , inaccordance with an illustrative embodiments.

As described in U.S. Pat. No. 9,272,280, which is incorporated byreference herein in its entirety, in various embodiments, the consumablecartridge contains a lens assembly that focuses ultrasound energy withinthe sample that can be used to generate streaming and mixing. The lensassembly, or sound focusing assembly, is designed using a soft material,such as a thermoplastic elastomer 134 (previously referred to as 21), inconjunction with a rigid substrate 132 (e.g., formed of testing block 21d), such as polystyrene as shown in FIGS. 10, 11, and 12 . Thiscombination provides a dry ultrasound coupling that does not require theuse of any fluid or gel couplant. Note that the same lens and ultrasounddriver used for hemostasis measurement can be used in this matter toprovide mixing. Increasing acoustic energy for mixing can be deliveredby, for example, increasing pulse length, pulse amplitude or pulserepetition frequency.

Referring now to FIG. 10 , a top cross-sectional view of the testingchamber 116 (referred to previously as testing chamber 16) is shown. Toseal each test chamber, e.g. test chamber 116, a lens assembly 131includes a rigid substrate 132 and a couplant 134 that can be positionedat the back end of each test chamber.

Referring still to FIG. 10 , each couplant 134 comprises an elastomericmaterial. Optionally, the elastomeric material is a thermoplasticelastomer (TPE). Example elastomeric materials optionally include,Dynaflex D3202, Versaflex OM 9-802CL, Maxelast 54740, RTP 6035,Versaflex CL2003X, among others. Optionally the couplant is over-moldedto the rigid substrate. Optionally the couplant is mechanically anchoredto the rigid substrate.

Referring still to FIG. 10 , between each couplant 134 and the openspace of each test chamber is a rigid substrate 132. The rigid substrateand the couplant form an interface that focuses ultrasound transmitted(e.g. lens assembly) by an ultrasonic transducer into the chamber's openspace and onto any biological fluid and/or reagents in the chamber. Therigid substrate of the lens can comprise a material which allows soundto pass and that can act to focus ultrasound at some level within thespace. Optionally, the rigid substrate comprises a styrene.

Referring now to FIG. 11 , The lens assembly may be glued or welded tothe surface 101 of the testing block 21 d (shown in FIG. 11 as element132) to secure the lens in place in an orientation that allows thedesired focusing of sound. Alternatively, the lens assembly isoptionally manufactured together with the surface 101 of the testingblock 21 d. In this regard, the rigid substrate 132 can be molded withthe surface 101 of the testing block 21 d and the couplant 134 can beovermolded or mechanically anchored on the rigid substrate. A widevariety of materials can be used to construct the device. For example,plastics can be used for single use, disposable cartridges.

Referring still to FIG. 11 , each of the test chambers 116 can have alens assembly positioned over the large opening of each chamber's openspace. In this way, each chamber can be separately interrogated byfocused ultrasound.

Referring still to FIG. 11 , when placed in the instrument, the couplant134 can be placed in acoustic communication with a transducer forsupplying ultrasound through the lens assembly and into a test chamber116. Optionally, an intermediate layer of an acoustically permeablematerial is positioned between an ultrasonic transducer and thecouplant. For example, and intermediate layer or block of Rexolite® orTPX® can be used. The intermediate layer can be forced against thecouplant and can be in acoustic contact with the transducer.

Referring still to FIG. 11 , sound generated by a transducer passesthrough the intermediate layer, through the couplant, through the rigidsubstrate, and is focused within the biological sample, such as blood,and reagent in the test chamber. Some of the sound directed into chambercontacts the distal interior surface 111 of the test chamber, which isdefined by the surface 126. Optionally, the surface is polystyrene. Thedistal interior surface has a known geometry and is positioned at aknown distance from the ultrasound source. The distal interior surface111 is used as a calibrated reflector, which is used to estimate thespeed of sound and attenuation of sound in a test chamber at base lineand during the process of clot formation and clot dissolution. Thesemeasurements can be used, for example, to estimate hematocrit of thesubject along with the indexes of hemostasis. The sound generated by thetransducer can be focused within the biological sample in a test chamberusing a parabolic mirror that is coupled to the biological sample usingan elastomer.

Other example cartridge apparatus and measurement system, and methodsthereof, are described in U.S. Pat. No. 9,031,701; U.S. ProvisionalAppl. No. 61/443,084; U.S. Pat. Nos. 9,272,280; 9,410,971; U.S.Provisional Appl. No. 61/443,088; U.S. Publication No. 2011/0252352;published PCT Publication No. WO2011/127436; U.S. Publication No.2012/0294767; U.S. Pat. Nos. 7,892,188; 8,740,818; and U. S. PublicationNo. 2016/0274067, each of which is incorporated by reference herein inits entirety.

As noted, the cartridge and features described herein can be modifiedfor use with other types of measurement systems such asthromboelastography-based systems, thromboelastometry-based systems,optical-based systems, fluorescence-based systems, colorimetric-basedsystems, aggregometry-based systems, resonance-based system, and anelectrical impedance-based system, among others.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which thisinvention pertains having the benefit of the teachings presented in theforegoing description. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

As used in the claims, the term “first”, “second”, and “third” areprovided merely as labels and do not intended to connote a sequence.

1. An apparatus comprising: a housing; an input port integrally formedwith the housing that is structurally configured to receive contents ofa sample holding tube; and a first chamber in fluidic communication withthe input port, the first chamber being configured as a heating chamberto receive and retain a sample contained in the sample holding tube andto condition the received sample to, or near, a desired temperaturebefore the received sample is allowed to contact one or more reagentslocated in two or more fluidic circuits downstream to the first chamber;the two or more fluidic circuits, including a first fluidic circuit anda second fluidic circuit, wherein each of the two or more fluidiccircuits comprises i) a second chamber in fluidic communication with thefirst chamber that meters the sample in the first chamber into analiquot, wherein the metered sample is introduced to a reagent, or acombination of reagents, located in a corresponding fluidic circuit toform a mixed sample and ii) a testing chamber in fluidic communicationwith the second chamber, the testing chamber being structurallyconfigured for interrogation by a measurement system configured todetermine at least one viscoelastic property of the mixed sample, andwherein the second chambers of the first fluidic circuit and the secondfluidic circuit are configured, via a controller, to be filled inparallel.
 2. The apparatus of claim 1, wherein at least one of the oneor more fluidic circuits comprise one or more pockets configured tohouse at least one lyophilized bead comprising the reagent, or thecombination of regents.
 3. (canceled)
 4. The apparatus of claim 1,wherein the input port is communicatively coupled to a first pressureport, wherein pressure when applied to the first pressure port causesthe evacuation of the contents of the sample holding tube through theinput port to the first chamber, wherein the input port forms a part ofa needle assembly structurally configured to establish fluidiccommunication and evacuate contents of the sample holding tube. 5.(canceled)
 6. The apparatus of claim 4, wherein the needle assemblycomprises the input port and a second port, wherein the second port isconfigured to vent a liquid or gas into the sample holding tube so as topromote evacuation of the contents therein.
 7. (canceled)
 8. Theapparatus of claim 1, wherein the first chamber is configured to matewith a corresponding thermal regulating surface of a sub-systemcomponent of the measurement system to condition the received sample to,or near, the desired temperature, and wherein the first chambercomprises a shape and/or material optimized to facilitate heating and/orcooling of the sample to, or near, the desired temperature. 9.(canceled)
 10. The apparatus of claim 8, wherein a channel portion ofthe one or more fluidic circuits is configured to mate with acorresponding thermal regulating system of the measurement system tocondition the received sample to the desired temperature, and whereinthe first chamber and/or the channel portion of the one or more fluidiccircuits is in physical proximity with a sensor of the measurementsystem configured to measure a temperature of the sample received in thefirst chamber.
 11. (canceled)
 12. The apparatus of claim 4, comprising afilter positioned within the first pressure port, wherein the filter isconfigured to allow air to move through the first pressure port butprevent fluid from moving there through.
 13. (canceled)
 14. Theapparatus of claim 1, wherein each of the second chambers is connectedto a second pressure port, wherein pressure when applied to the secondpressure port causes the filling of the second chamber, and wherein eachof the test chambers is connected to a third pressure port, wherein whenpressure is applied to the third pressure port causes the test sample toflow toward the test chamber through a respective serpentine conduit.15. The apparatus of claim 14, wherein each of the second chambers isconnected to a vent port, wherein the vent port is configured to beclosed while the sample is metered into the aliquot in the secondchamber and further configured to be open to atmospheric pressure afterthe sample is metered into the aliquot in the second chamber.
 16. Theapparatus of claim 1, wherein each of the one or more fluidic circuitscomprises a third set of fluid pathways in fluidic communication betweena respective second chamber and test chamber, wherein a portion of thirdset of fluid pathways is arranged as a serpentine-shaped conduit,wherein the serpentine-shaped conduit forms a serpentine reservoirbetween the testing chamber and the second chamber, and wherein ametered sample is directed through portions of the serpentine-shapedconduit to facilitate mixing of the metered sample and the reagent, orthe combination of reagents.
 17. The apparatus of claim 16, wherein themetered sample is alternatively and multiplicatively directed, for eachof the one or more fluidic circuits, between a first position in afluidic circuit a second position in the fluidic circuit, wherein thelength first position and second portion includes at least a portion ofthe serpentine-shaped conduit.
 18. (canceled)
 19. (canceled)
 20. Theapparatus of claim 16, wherein the serpentine-shaped reservoir includesan optical detection zone.
 21. The apparatus of claim 1, wherein each ofthe one or more fluidic circuits further comprises a mixing zone betweenthe testing chamber and the second chamber, the mixing zone comprisingone or more ferromagnetic beads or bars therein.
 22. The apparatus ofclaim 1, wherein at least one of the one or more fluidic circuitscomprises one or more quality testing portals, wherein the one or morequality testing portals is configured to be sensed optically orelectrically to sample for characteristics of the metered sample. 23.(canceled)
 24. The apparatus of claim 1, wherein the testing chambercomprises a lens configured to direct ultrasonic pulses generated by themeasurement system into the testing chamber.
 25. (canceled)
 26. Theapparatus of claim 1, wherein at least one of the reagents, orcombination of reagents, located in the one or more fluidic circuits isselected from the group consisting of an intrinsic pathway activator, anextrinsic pathway activator, a coagulation activator, plateletactivator, a platelet inhibitor, a fibrinolytic function inhibitor,thrombomodulin, polybrene, heparin, corn trypsin inhibitor, adenosine,calcium, and heparinase I, or a combination thereof.
 27. (canceled) 28.(canceled)
 29. The apparatus of claim 1, wherein the measurement systemis selected from the group consisting of a sonorheometry-based system,thromboelastography-based system, a thromboelastometry-based system, anoptical-based system, a fluorescence-based system, a colorimetric-basedsystem, an aggregometry-based system, a resonance-based system, and anelectrical impedance-based system.
 30. The apparatus of claim 1,comprising: at least four test channels, wherein a first test channelcomprise an intrinsic pathway activator, wherein a second test channelcomprises the intrinsic pathway activator and a heparin neutralizer,wherein a third test channel comprises an extrinsic pathway activator,and wherein a fourth test channel comprises the extrinsic pathwayactivator and a platelet inhibitor.
 31. The apparatus of claim 1,comprising: at least four test channels, wherein a first test channelcomprise an intrinsic pathway activator, wherein a second test channelcomprises an extrinsic pathway activator and a fibrinolytic functioninhibitor, wherein a third test channel comprises an extrinsic pathwayactivator, and wherein a fourth test channel comprises the extrinsicpathway activator and a platelet inhibitor, wherein the third channeland the fourth channel each includes Hexadimethrine bromide (polybrene),and wherein the fourth test channel further includes a fibrinolyticfunction inhibitor.
 32. The apparatus of claim 30, wherein the thirdchannel and the fourth channel each includes Hexadimethrine bromide(polybrene). 33.-44. (canceled)